Poliovirus is an enterovirus, a gene of the family Picoronaviridae. The structure of the poliovirus is known, and is highly conserved among strains and serotypes.
The polio virus genome exists as a single-stranded RNA molecule of approximately 7,500 nucleotides. The RNA is comprised of three regions; a 5'untranslated region of 743 nucleotides, an open reading frame of 6,618 nucleotides, and a 3'untranslated region of 72 nucleotides followed by a terminal poly (A) tail (Refs 1, 2--various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure). The open reading frame can be further subdivided into the P1, P2 and P3 regions. The genes found in the P1 region encode for the capsid proteins. These genes can be deleted without effecting the ability of the viral RNA to translate or replicate (ref. 3). The P2 and P3 regions (encoding the non-structural proteins) (ref. 1) and features of the 5' untranslated region are essential for replication and translation (refs. 4, 5, 6, 7).
The ability to remove the structural P1 genes from the viral genome without compromising the ability of the RNA to replicate has been used both to study the mechanisms of replication and encapsidation (refs. 4, 5) and to produce foreign proteins whose genes have been placed in the P1 position. These include various Human Immunodeficiency Virus proteins (refs. 8, 9, 10), Simian Immunodeficiency Virus proteins (ref. 11) and carcinoembryonic antigen (ref. 12).
Most studies of polio virus (including those mentioned above) have been carried out by transfecting cells with RNA synthesised in vitro using bacteriophage T7 RNA polymerase (ref. 13). The reasoning behind this activity has been that polio virus does not, during a normal infection, enter the nucleus, and the RNA does not, therefore, undergo splicing or other nuclear processing events. It has been suggested that the polio RNA sequence contains adventitious splicing and polyadenylation signals which could negatively affect the RNA (ref. 13). Further, since the RNA would be embedded as part of a potentially larger primary transcript, biologically active, replication competent molecules may not be produced (ref. 13). In contrast, one early report demonstrated that infectious polio virus particles could be obtained by transient transfection of cell lines with plasmids harbouring cDNA copies of the polio virus genome (ref. 14). It is, however, unclear whether a cDNA containing the coding sequence for a foreign protein in place of the polio structural genes will generate an exportable transcript capable of replication when positioned behind a mammalian promoter. Although it is possible to construct replicons from RNA harbouring foreign genes, this system has the disadvantage that it is difficult to accurately check the sequence which has been transfected into cells, given the inherent infidelity of the RT-PCR reaction. This can be a particular concern when the proteins to be produced are to be subsequently used for human vaccination.
Transfection of cells with DNA encoding the polio replicon harbouring a foreign gene would allow for easy recovery and confirmation of transfected sequences. Moreover, DNA is inherently more stable and easier to work with than RNA. Finally, self replicating RNAs may be particularly useful when examining the regulation of a weak promoter. The potential problems of producing quantities of potentially toxic proteins, including the non-structural proteins of polio, could be overcome by the use of a suitably tight and inducible mammalian promoter system. Coupled to such a promoter, the polio replicon system may allow for rapid and high level increases in protein amounts post induction.